Epidemic cholera caused by toxigenic Vibrio cholerae O1 is a major health problem in several developing countries. Traditional methods for identifying V. cholerae involve cultural, biochemical and immunological assays which are cumbersome and often take several days to complete. In the present study, a direct cell multiplex PCR was developed targeting the ompW, ctxB and rfbO1 genes for confirmation of V. cholerae, its toxigenicity and serogroup Ol, respectively from clinical and environmental samples. The detection sensitivity of the multiplex PCR was 1.9 x 103V. cholerae per PCR reaction. A total of 31 environmental samples and 45 clinical V. cholerae isolates from different outbreaks were examined by the PCR. The assay was simple and specific, as there was no requirement for DNA extraction and no amplification was observed with other homologous bacteria used. The assay can be very useful for rapid surveillance of toxigenic V. cholerae O1 in environmental water samples, as well as for confirmation of clinical isolates.